Simultaneous Determination of Free, Total, and Normalized Concentrations of Bioactive Compounds Medication is the most relied-upon treatment in health care today; however, the current drug-use system suffers from problems related to insufficient dose individualization and adverse reactions. In order to better understand therapeutic and toxic effects of bioactive compounds and to continue developing more selective and effective treatments, comprehensive analysis of biological samples is growing in importance. Furthermore, the need for fast and affordable medical diagnostics is experiencing a growing trend. Current chemical analysis protocols completely overlook plasmatic proteins and lipoproteins that bind the investigated bioactive compound; these proteins can significantly change a chemical?s pharmacokinetic profile and optimal concentration range (expressed as free concentration). When clinical samples are analyzed only for total concentrations, important information regarding compound-binding proteins and free concentrations is irreversibly lost. Plasma protein binding affects the amount of compound available for diffusion into target tissues, the calculation of in vivo hepatic clearance, and the interpretation of bioavailability. One of the best approaches for optimizing therapeutic regimens consists of monitoring the compound?s concentration in blood. In addition to its well established role in drug development, pharmacokinetic monitoring is increasingly being used in clinical practice to guide dosage regimens. However, measurement of the total concentrations does not provide the needed information concerning the unbound concentration of bioactive compound which is available for pharmacodynamic action. To address this, a normalized concentration can be determined based on the observed total concentration and the serum protein concentration. The purpose of the proposed research is to develop validated approaches and equations for determining free, total, and normalized concentrations for several model bioactive compounds (starting with hormones). These equations will allow for personalized therapy (precision medicine) by providing the means to individualize dosage regimens according to each patient?s blood composition. The study will be carried out in two specific aims focused on: ? Determine normalized levels of testosterone and thyroid hormones in samples from healthy donors a. Adapt our existing microextraction methods to measure hormones in biological samples b. Determination of individual-specific plasma binding capacity for the investigated compounds ? Validate the use of normalized concentrations of hormones in patients a. Generation of compound-specific equations for normalized concentrations b. Establish correlations with markers of health status in patients